Uracil is a normally-occurring base in RNA. However, uracil may also be found in DNA due to deamination of cytosine or misincorporation of dUTP during DNA replication. Because uracil will base-pair with adenine, deamination of cytosine results in a transition mutation from G:C to A:T. All known DNA-containing organisms therefore have a specific repair mechanism for removing uracil from DNA. The enzyme uracil DNA glycosylase (UDG) catalyzes the first step of the repair pathway, in which the N-glycosidic bond between uracil and the deoxyribose sugar is hydrolyzed. Other enzymes then repair the abasic site. UDG may also be referred to as uracil N-glycosylase (UNG). The UDG enzyme is highly specific for removal of uracil in DNA. It does not have any detectable excision activity against uracil in RNA (which could potentially inactivate mRNA, rRNA and tRNA in the cell) nor does it have any detectable activity against thymine in DNA (which would compromise the structure and stability of chromosomal DNA).
The UDG enzyme of the mesophilic organism Escherichia coli has been studied the most extensively, and the gene encoding the protein (ung) has been cloned (U. Varshney, et al. 1988. J. Biol. Chem. 263:7776-7784). The UDG genes of herpes simplex virus type-1, Haemophilus influenzae (1995. Science 269(5223):496-512), Streptococcus pneumoniae (1990. Nucl. Acids Res. 18(22):6693) and Bacillus subtilis (1993. Mol. Microbiol. 10(2):371-384) have also been isolated and sequenced. Thermophilic UDG proteins have been isolated from the thermophilic bacteria Bacillus stearothermophilus and Thermothrix thiopara, which have optimum temperatures for growth of 55.degree. C. and 75.degree. C., respectively (O. K. Kaboev, et al. 1981. FEBS Lett. 132:337-340; O. K. Kaboev, et al. 1985. J. Bacteriol. 164:421-424). The UDG genes are fairly homologous. However, in spite of sequence divergence demonstrated by hybridization studies and sequence analysis, the tertiary structure of the UDG enzymes has been found to be highly conserved (U. Varshney, et al., supra).
Nucleic acid amplification reactions are processes by which specific nucleic acid target sequences are amplified. Amplification methods have become powerful tools in nucleic acid analysis and preparation and several nucleic acid amplification methods are known. These include the Polymerase Chain Reaction (PCR), Self-Sustained Sequence replication (3SR), Nucleic Acid Based Sequence Replication (NASBA), the Ligase Chain Reaction (LCR), Q.beta. replicase amplification and Strand Displacement Amplification (SDA). Unfortunately, the powerful ability of these nucleic acid amplification methods to amplify minute quantities of a target sequence also make them susceptible to contamination by copies of target sequences (amplicons) which may be carried over from previous amplification reactions in reagents, pipetting devices and laboratory surfaces. These contaminating products of previous amplifications may themselves be amplified in a subsequent amplification reaction. Even a few molecules of a contaminating target sequence may be readily amplified and detected, resulting in falsely positive results.
Recently developed methods for inactivating contaminating amplicons in nucleic acid amplification reactions such as PCR and SDA involve incorporation of the nucleotide deoxyuridine triphosphate (dUTP) into amplified nucleic acid sequences in place of thymidine triphosphate (TTP). As deoxyuridine (dU) is rarely found in naturally-occurring DNA, this nucleotide serves to distinguish previously produced amplicons from new target sequences which have not yet been amplified. The uracil-containing DNAs, representing previously amplified contaminating sequences, are treated with UDG to remove the intentionally incorporated uracil in amplified nucleic acid. Uracil is removed without destruction of the sugar-phosphodiester backbone, thereby producing an abasic site in the DNA. These abasic sites are susceptible to hydrolysis by heat or alkali, a process which fragments the uracil-containing DNA and renders it unamplifiable in a subsequent nucleic acid amplification reaction. UDG is inactivated prior to beginning the subsequent amplification reaction to prevent removal of uracil residues from newly generated amplicons. Fragmentation of the nucleic acids is optional, as it has been found that the abasic sites alone are sufficient to prevent amplification.
As E. coli UDG has been the preferred enzyme for decontamination of nucleic acid amplification reactions, it is typically inactivated by incubation at high temperatures (70.degree.-80.degree. C.). In fact, mesophilic UDG's are substantially inactive at the elevated temperatures used for PCR amplification reactions. However, it has been shown that upon return of the PCR sample to 4.degree.-25.degree. C. after amplification, sufficient UDG activity is still present to degrade dU-PCR amplification products. It has therefore been recommended that PCR reactions be maintained at elevated temperatures after UDG treatment (Rashtchian, A., Hartley, J. L. and Thornton, C. G., Biotechniques, volume 13, No. 2, page 180). To address the problem of residual UDG activity after heat inactivation, WO 92/01814 describes a thermolabile UDG enzyme.
The Bacillus subtilis bacteriophage PBS1 is unique in that it uses uracil instead of thymine in its DNA. This phage must therefore protect itself from host cell UDG's upon infection. To do so, PBS1 produces a uracil glycosylase inhibitor protein (UGI) which complexes with UDG's and inactivates them. The PBS1 gene encoding UGI, and genes from closely related B. subtilis PBS phages such as PBS2, have been cloned and expressed to produce recombinant UGI. The UGI protein has been shown to be an effective means for controlling residual UDG activity still present after heat inactivation in PCR (Rashtchian, et al., supra). It has further been shown that UGI alone is effective to inactivate UDG in isothermal amplification reactions such as Strand Displacement Amplification (SDA), which do not have high temperature cycling and which may be incompatible with high temperature steps for inactivation of UDG.
As previously stated, genes coding for mesophilic UDG's have been cloned and expressed to produce recombinant UDG's for use in decontamination of nucleic acid amplification reactions. However, temperature cycling amplification reactions such as PCR are typically performed at thermophilic temperatures and it is advantageous to perform isothermal amplification reactions such as SDA at thermophilic temperatures to improve the speed and specificity of the reaction. Such thermophilic amplification reactions would benefit from the availability of a thermostable UDG which would be active at the preferred temperature of the amplification reaction. Such a thermophilic UDG would permit the practitioner to perform decontamination at a higher temperature which may be similar to or the same as the thermophilic amplification reaction (e.g., 45.degree.-75.degree. C.). This makes the steps of the process more efficient, as the magnitude of the temperature changes required to decontaminate the sample, increase temperature to inactivate the UDG, and re-equilibrate the sample to the reaction temperature is significantly reduced as compared to decontamination at mesophilic temperatures (e.g., 35.degree.-45.degree. C.).